The effect of spherical aberrations on images. The effect of spherical aberrations on images. Mitotic spindles of HeLa cells were stained for microtubules (green), centrosomes (red), and chromosomes (blue). Z-stacks of images were acquired using a U Plan-Apo 100×/1.35 NA objective on a DeltaVision microscope. Deconvolution and volume rendering were performed using SoftWoRx software. A–C show projections viewed from the xy axis (the view seen down the microscope) and D–F viewed from the z axis (rotated around the y-axis by 90°). Here I used oils of different refractive index (r.i.) to demonstrate the spherical aberrations caused by r.i. mismatch between lens immersion medium and sample. Using the correctly matched oil for this sample (B and E; r.i. 1.518), the spindle fibers and centrosomes are cleanly resolved even in the z-axis view (E). Using mismatched oils (r.i. 1.504 or 1.530), some effects on the xy images can be seen (A and C; note the more blurred spindle fibers) but the z-axis views are more markedly compromised (D and F), with elongated, blurred structures and streaks fanning out toward the edges. Alison J. North J Cell Biol 2006;172:9-18 © 2006 Rockefeller University Press